In Phase I, the cviPIIM, cviPIINt, cviQXIM, cviQXINt genes were cloned and sequenced. Recombinant Nt.CviPII was expressed in E.coli and purified. Nt.CviQXI was expressed in E.coli and by an in vitro transcription/translation system. Nt.Sapl and Nb.Sapl were engineered from Sapl endonuclease using a novel two-step genetic selection procedure. Under Phase II funding, we will optimize the expression conditions for Nt.CviPII and Nt.CviQXI to alleviate cytotoxicity. Constitutive expression of M.CviPII and M.CviQXI will be achieved to fully modify host DNA. Nt.CviPII and Nt.CviQXI NEases will be expressed in pACYC-T7. In addition, C-terminal truncation variants of Nt.CviPII and Nt.CviQXI will be constructed (in fusion with intein and chitin binding domain). Synthetic peptides will be ligated to the truncated versions via intein-mediated peptide ligation to construct full-length enzymes. Secondary amino acid (aa) substitutions will be introduced into Nb.Sapl to optimize the nicking activity and eliminate dsDNA cleavage. The equivalent aa changes will be introduced into BspQI (a thermostable Sapl isoschizomer) to isolate N.BspQI. Sapl and Earl share similar DNA recognition sequences and the aa sequences of the two endonucleases display a high level of similarity. The aa changes that resulted in Sapl nicking variants will be introduced into Earl endonuclease to isolate N.Earl. We will isolate nicking variants from a 4-base cutter Mnll (CCTCN7 A, ANeGAGG). Extremely thermostable nicking variants will also be isolated from Tth111II (CAARCA11/9) that is active at 75 degrees C. We will engineer nicking variants from homing endonucleases l-Scel and l-Dmol with15-18 bp recognition sequences. We will clone and characterize HNH type homing endonucleases from chlorella virus genomes. In Phase I, we discovered a novel DNA amplification method using Nt.CviPII and Bst DNA polymerase. Random DNA amplification has been achieved from a small amount of genomic DNA or from a single bacterial colony without addition of exogenous primers. Under Phase II funding, this isothermal amplification method (NEMDA) will be optimized and the sensitivity will be improved. Finally, we will investigate the potential of NEMDA for detections of pathogens and its use in bio-prospecting.